Use of lupus la protein as a target for antibody diversity regulation

Abstract

The application belongs to the technical field of biology, and discloses application of Lupus La protein as an antibody diversity regulation target point. The application is D1 or D2: D1, application of Lupus La protein (La) or genetic material thereof in improving the efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci; D2, application of a substance for knocking out a La protein coding gene or a substance targeting the La protein coding gene in reducing the efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci. The application first discovers that the La protein can be used as a structural adapter factor and can directly mediate AID targeting immunoglobulin heavy chain loci, thereby filling the blank of a mechanism of "non-transcription-dependent spatial positioning of AID". The expression level of the protein of the application is linearly positively correlated with CSR / SHM activity, and the protein can be used as an immune enhancement target point, a risk marker and an inhibition target point.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to the application of Lupus La protein as a target for antibody diversity regulation. Background Technology

[0002] Antibody diversity is fundamental to the adaptive immune system's recognition and clearance of pathogens. During B lymphocyte development and activation, immunoglobulin genes undergo key molecular events such as V(D)J rearrangement, class switch rearrangement (CSR), and somatic hypermutation (SHM), resulting in antibody subtypes with different effector functions and antigen affinities. CSR achieves the conversion from IgM to isotypes such as IgG, IgA, or IgE by replacing the constant region (C region) of the immunoglobulin heavy chain, while SHM introduces point mutations in the V(D)J exon region of germinal center (GC) B cells, providing a structural basis for affinity maturation. Both processes are initiated by activation-induced deaminase (AID). AID introduces a U:G mismatch into the target sequence through deoxycytidine deamination, triggering DNA repair and rearrangement pathways, thereby achieving antibody gene rearrangement or mutation.

[0003] Although AID expression has been confirmed as a prerequisite for CSR and SHM, how it is specifically directed to immunoglobulin heavy chain (IgH) loci in the genome, rather than other transcriptionally active regions, remains a key unsolved scientific question. Existing research indicates that transcriptional activity is a necessary but not sufficient condition for AID targeting, suggesting the existence of additional targeting regulators. Several proteins interacting with AID have been reported (such as RPA, Spt5, and PTBP2), which are mainly involved in maintaining AID stability, transcriptional coupling, or nuclear localization. Furthermore, chromatin structural proteins (such as CTCF and the Cohesin complex) indirectly affect AID accessibility by maintaining the spatial conformation of immunoglobulin heavy chain loci. However, structural adaptors that directly mediate AID-specific targeting of immunoglobulin heavy chain loci (rather than other transcriptionally active regions) remain to be identified. In addition, although specific regulatory elements in immunoglobulin heavy chain loci, such as the 3′RR regulatory sequence, have been shown to have enhancer activity, there is still a lack of direct evidence as to whether they directly participate in the targeting mechanism of AID or only indirectly affect AID activity by promoting transcription.

[0004] The lack of understanding of these mechanisms leads to significant limitations in current intervention methods for AID-related functional impairments. Specifically: (1) In the treatment of CSR deficiency-related diseases, for class switching and recombination disorders caused by AID dysfunction (such as some high IgM syndromes), the main clinical treatment relies on intravenous immunoglobulin (IVIG) replacement therapy. Although this regimen can supplement functional antibodies, it has the following technical drawbacks: it requires lifelong administration, the treatment cost is high, and it can only replace antibody function, but cannot restore the AID's targeting ability at the immunoglobulin heavy chain locus. It is a functional replacement rather than etiological repair. Although hematopoietic stem cell transplantation has the potential for radical cure, its clinical application is limited by factors such as donor availability, age, and transplant-related complications.

[0005] (2) In the treatment of B-cell lymphoma, the abnormal expression and spatial mislocalization of AID have been confirmed as an important pathogenic mechanism of some B-cell lymphomas (such as diffuse large B-cell lymphoma). Existing treatments (including chemotherapy, small molecule targeted drugs and immunotherapy) mainly work by inhibiting tumor cell proliferation or inducing apoptosis, but have not been able to directly correct the abnormal spatial localization of AID in cells, and therefore it is difficult to block tumor progression at the pathogenic mechanism level.

[0006] (3) Regarding enhanced vaccine response, for low vaccine response due to insufficient AID targeting efficiency (common in some immunodeficient individuals and the elderly), existing adjuvants mainly enhance immunogenicity by activating inflammatory pathways. Although some adjuvants can indirectly improve germinal center response, there is currently no technology that can directly improve the targeting efficiency of AID at immunoglobulin heavy chain loci, making it difficult for the above-mentioned populations to obtain effective protection through existing vaccine strategies.

[0007] Currently accepted AID targeting models all rely on transcription processes and related structures, primarily focusing on explaining the generation of AID substrates (ssDNA), but failing to elucidate how AID is specifically recruited to immunoglobulin heavy chain sites: The R-loop model (Alt lab, Nature Immunology, 2003; Lieber lab, Nature Immunology, 2003): Transcription products form RNA-DNA hybrids with template DNA, exposing ssDNA as the AID substrate. However, this model only describes how the substrate is generated, without revealing how AID is specifically recruited to immunoglobulin heavy chain sites, nor does it provide any controllable recruitment factors. The transcriptional pausing model (Nussenzweig lab, Cell, 2010): RNA polymerase II pauses in the S region, increasing the AID action window, but this is still a transcription-dependent mechanism and cannot explain why only immunoglobulin heavy chains are efficiently targeted by AID among transcriptionally active genes. Anisotropic / bidirectional transcription models (Basu lab, Nature, 2014; Alt lab, Cell, 2014): These models promote AID targeting through anisotropic / bidirectional transcription producing non-coding RNA or topological changes, but they still rely on the transcription process itself and cannot explain the specific selection of AID for the immunoglobulin heavy chain. G-quadruplex (G4) structures (Chaudhuri lab, Cell, 2015): Transcription-dependent G4 structures recruit AID, but this is still a "transcription-structure coupling" mechanism and fails to provide specific determinants independent of transcription. Limitations of 3′RR function studies: Vincent-Fabert et al. (Blood, 2010) demonstrated the indispensability of the 3′RR segment for CSR by systemic deletion of 30-kb immunoglobulin heavy chain 3′RR; however, this study only reached the level of "DNA segment functional necessity," without revealing which specific factors mediate the physical recruitment of AID to 3′RR, let alone clarifying whether this recruitment is independent of transcription.

[0008] In summary, there is an urgent need in this field to identify and validate manipulable molecules that can directly mediate AID targeting to immunoglobulin heavy chain loci, in order to provide new targets and strategies for the treatment of AID-related diseases, enhancement of vaccine responses, and mechanistic intervention in B-cell lymphoma. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide a structural adaptor factor that can directly mediate AID targeting the immunoglobulin heavy chain gene locus, thereby filling the gap in the existing technology in regulating antibody diversity and solving the technical problems of insufficient CSR (immunodeficiency, low vaccine response) and AID off-target (genomic instability, lymphoma) caused by AID targeting defects.

[0010] To address the aforementioned technical issues, this invention, through in vitro and in vivo experiments, has for the first time discovered that the Lupus La protein (La) can act as a structural bridge, specifically binding to the immunoglobulin heavy chain locus regulatory region and directly recruiting AID to the CSR / SHM occurrence site, forming an "AID-La-immunoglobulin heavy chain" functional complex, thereby promoting the precise targeting of AID in a manner independent of transcriptional regulation.

[0011] Based on the above findings, the present invention provides the following technical solution: Application of Lupus La protein or related biomaterials of Lupus La protein in D1 or D2: The use of D1, Lupus La protein or related genetic material in improving the efficiency of activation-induced cytidine deaminase (AID) targeting immunoglobulin heavy chain loci, or in the preparation of drugs that improve the efficiency of activation-induced cytidine deaminase (AID) targeting immunoglobulin heavy chain loci. The genetic material is at least one of the following: c1. The nucleic acid molecule encoding the Lupus La protein; c2, expression cassettes, recombinant vectors, recombinant microorganisms, transgenic cell lines, transgenic animal tissues or transgenic organs containing the nucleic acid molecules described in c1; D2. The use of substances that knock out the Lupus La protein-coding gene or substances that target the Lupus La protein-coding gene in reducing the efficiency of activation-induced cytidine deaminase (AID) targeting the immunoglobulin heavy chain locus, or in the preparation of drugs that reduce the efficiency of activation-induced cytidine deaminase (AID) targeting the immunoglobulin heavy chain locus.

[0012] The Lupus La protein of this invention is a multifunctional RNA-binding protein encoded by the SSB (Sjoegren Syndrome Antigen B) gene, with a molecular weight of approximately 47 kDa, playing a central role in RNA metabolism. The La protein is an evolutionarily conserved RNA-binding protein with a typical modular structure, consisting of an N-terminal winged helix-turn-helix (wHTH, i.e., the La motif), a central RNA recognition motif (RRM), a C-terminal atypical variant RNA recognition motif (xRRM), and highly disordered regions.

[0013] In some embodiments, the Lupus La protein is a protein of type A1, A2, or A3: A1. The amino acid sequence is the protein that is the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 in the sequence listing; A2. A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 in the sequence listing, which has more than 90% sequence identity with the protein shown in A1 and has the characteristics of A2.1-A2.4. A2.1. It has a regulatory region that binds to immunoglobulin heavy chain loci; A2.2, It directly interacts with the activation-induced cytidine deaminase AID protein; A2.3, mediates the spatial targeting of AID in the immunoglobulin heavy chain locus transition region or variable region; A2.4. It has the function of promoting antibody class switching recombinant CSR and somatic high-frequency mutant SHM; A3, a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1 or A2.

[0014] In the above applications, SEQ ID No. 1 in the sequence listing consists of 415 amino acid residues, and SEQ ID No. 2 consists of 408 amino acid residues.

[0015] In the above applications, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0016] In the above applications, the 90% or more of identity can be at least 90%, 95%, 98%, or 99% identity.

[0017] In the above applications, the nucleic acid molecule mentioned in c1 can specifically be a nucleic acid molecule whose coding sequence is SEQ ID No. 3 in the sequence listing.

[0018] In the above applications, the efficiency of the activation-induced cytidine deaminase targeting the immunoglobulin heavy chain locus is the antibody type switching rearrangement (CSR) ratio and / or somatic high-frequency mutation (SHM) frequency.

[0019] In some embodiments, the antibody type switching rearrangement ratio is the IgA class switching rate, the IgG1 class switching rate, and / or the IgG3 class switching rate.

[0020] In one embodiment, the IgA class conversion rate is the rate of conversion from IgM to IgA, the IgG1 class conversion rate is the rate of conversion from IgM to IgG1, and the IgG3 class conversion rate is the rate of conversion from IgM to IgG3.

[0021] In the above applications, the drug described in D1 that improves the efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci can be a drug that enhances vaccine response, or a drug for treating diseases caused by antibody type switching rearrangement defects.

[0022] Antibody class-switch rearrangement (CSR) deficiency refers to a group of diseases in which B lymphocytes are unable to convert antibodies from IgM to other types such as IgG, IgA, or IgE during the immune response, resulting in immune dysfunction. An example is hyper-IgM syndrome.

[0023] The use of the drug described in D1, which enhances the efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci, is as follows: when a subject's Lupus La protein level is below a normal threshold (e.g., the 25th percentile) and is accompanied by antibody type switching rearrangement defects (e.g., high IgM syndrome), or when a subject has a low vaccine response, the drug is used to supplement Lupus La protein to promote antibody type switching rearrangement and antibody diversity by restoring the targeting ability of activation-induced cytidine deaminase (AID) at immunoglobulin heavy chain loci.

[0024] In some embodiments, the substance described in D2 for knocking out the Lupus La protein-coding gene is a CRISPR-Cas9 system containing gRNA and Cas9 targeting the Lupus La protein-coding gene.

[0025] In some embodiments, the substance targeting the Lupus La protein-coding gene described in D2) is a gRNA targeting the Lupus La protein-coding gene.

[0026] In some embodiments, the sequence of the gRNA targeting the Lupus La protein-coding gene is shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7.

[0027] In the above applications, the drug described in D2 that reduces the efficiency of activation-induced cytidine deaminase targeting the immunoglobulin heavy chain locus can be used to treat diseases with immunoglobulin heavy chain-Myc translocation.

[0028] The disease characterized by immunoglobulin heavy chain-Myc translocation is lymphoma or multiple myeloma. Specifically, the lymphoma may be B-cell lymphoma.

[0029] The use of the drug described in D2, which reduces the efficiency of activation-induced cytidine deaminase targeting the immunoglobulin heavy chain locus, is as follows: when a subject's Lupus La protein level is above a normal threshold (e.g., the 75th percentile) and accompanied by markers of genomic instability (e.g., immunoglobulin heavy chain-Myc translocation), the drug B is used to inhibit Lupus La protein expression or activity, thereby preventing or treating diseases with immunoglobulin heavy chain-Myc translocation by limiting AID off-target activity.

[0030] The present invention also protects a method for promoting antibody class switching rearrangement (CSR) and / or somatic high-frequency mutation (SHM) in B cells, comprising introducing the Lupus La protein or its encoding gene into target B cells to promote antibody class switching rearrangement and / or somatic high-frequency mutation in the target B cells.

[0031] This invention also protects recombinant B cells derived from target B cells, wherein the recombinant B cells are prepared by a method comprising the following steps: introducing the Lupus La protein or its encoding gene into target B cells to obtain recombinant B cells, thereby promoting antibody class switching rearrangement and / or somatic high-frequency mutation in the target B cells.

[0032] The target B cells are mammalian B lymphocytes (such as the CH12F3 cell line).

[0033] In some embodiments, the target B cell is an isolated cell.

[0034] The antibody type conversion rearrangement rate and / or somatic high-frequency mutation frequency of the recombinant B cells are higher than those of the target B cells.

[0035] This invention also protects a method for shortening the antibody development cycle, comprising: 1) providing cells expressing the Lupus La protein as described in any one of claims 1-3; 2) Contact the cells described in step 1) with antigen-specific B cells to enhance the somatic high-frequency mutation (SHM) frequency of the antigen-specific B cells, thereby shortening the antibody development cycle.

[0036] The present invention also protects a vaccine-adjuvant composition comprising Lupus La protein or a substance that increases the content or activity of said Lupus La protein, and a vaccine antigen.

[0037] This invention also includes a kit for diagnosis or prognostic assessment, the kit comprising an antibody or nucleic acid probe that specifically recognizes the Lupus La protein. The kit is used to differentiate between high and low CSR activity states, or for early screening and risk stratification of DLBCL.

[0038] The present invention also provides the application of the Lupus La protein or related biological materials as diagnostic biomarkers for immune diseases.

[0039] In this application, "increase" means a positive change of at least 10%, 25%, 50%, 75%, 100%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, or 800%.

[0040] In this application, "reduction" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0041] The method for verifying and regulating the function of the Lupus La protein (La) of this invention is applicable to various B cell models (including immortalized B cell lines, primary B cells, and in vivo animal models) and various pathogen infection and tumor immune response scenarios. Therefore, the La function and its bidirectional regulatory value verified by the method of this invention can be applied to various applications such as immunodeficiency disease treatment, vaccine adjuvant development, and lymphoma risk screening. The specific application scenarios and / or application prospects of this invention are as follows: 1. Suitable candidates for immunodeficiency treatment: patients with CSR deficiency (such as high IgM syndrome) and immunodeficiency populations due to insufficient antibody diversity. Application method: supplement La through lentiviral vectors or inducible expression systems to restore the targeting ability of AID at immunoglobulin heavy chain loci, fundamentally repair CSR function, and replace the existing lifelong IVIG maintenance therapy.

[0042] 2. Vaccine adjuvant development application scenarios: Vaccination enhancement mechanism for low vaccine response populations (elderly, immunosuppressed patients): Utilizing La to enhance AID targeting efficiency, improve CSR and SHM levels in germinal center response, accelerate affinity maturation, and enhance vaccine protective efficacy and durability.

[0043] 3. Application scenarios for prognostic assessment of infectious diseases: Clinical prognostic stratification detection methods for patients with acute infections such as COVID-19, influenza, and EB virus: Single-cell sequencing or ELISA to detect the expression level of La in patients' B cells. Clinical value: High La expression is positively correlated with effective CSR / antibody response, and can identify patients with poor prognosis at an early stage.

[0044] 4. Risk screening and intervention for B-cell lymphoma: Risk warning: By detecting La expression levels (above the 75th percentile) combined with genomic instability markers (γH2AX), the risk of developing lymphomas such as DLBCL can be predicted. Treatment intervention: La inhibitors or shRNA are used in high-risk populations to limit AID off-target activity and prevent malignant transformation caused by genomic instability.

[0045] 5. Optimization of application scenarios for in vitro antibody engineering: Therapeutic monoclonal antibody R&D technology route: Culture antigen-specific B cells on feeder cell layers expressing exogenous La, utilize La-mediated AID targeting to enhance SHM efficiency, shorten the affinity maturation cycle, and rapidly obtain high-affinity antibody candidates.

[0046] The core innovation of this invention lies in: Transcription-independent: La-mediated AID recruitment is independent of transcriptional pauses, R-loops, or G4 structures. Even at the same transcriptional level, La deletion completely abolishes AID targeting, demonstrating that its role is a specific determinant rather than an accessory effect of transcription.

[0047] Precise controllability: By regulating La expression or interfering with the binding of its HTH / RRM domain to 3′RR, AID targeting efficiency can be precisely controlled without affecting global transcription, providing a controllable molecular switch for antibody engineering; Dual physiological and pathological regulation: La levels not only determine the efficiency of physiological CSR / SHM (immune protection), but also directly regulate the off-target activity of pathological AID (lymphoma development), which is a precise regulation that existing transcription-dependent models cannot achieve.

[0048] The beneficial effects of this invention are as follows: 1) First time revealing structural targeting mechanism: This invention is the first to discover that La, as a structural aptamer, can directly mediate AID targeting immunoglobulin heavy chain gene loci, filling the gap in the mechanism of "non-transcription-dependent AID spatial localization" and providing a new molecular target for precise regulation of antibody diversity.

[0049] 2) Bidirectional regulatory value: This invention confirms that the expression level of La is linearly positively correlated with CSR / SHM activity. It can be used as an immune enhancement target (to treat CSR defects and enhance vaccine response), a risk marker (to provide early warning of genomic instability and lymphoma), and an inhibitory target (to prevent tumors caused by AID off-target effects), thus having unique bidirectional application value.

[0050] 3) Promising clinical translation prospects: Based on single-cell sequencing data analysis of human SARS-CoV-2 vaccine recipients, patients infected with multiple pathogens (including influenza A virus H1N1, SARS-CoV-2, EB virus, porcine circovirus type 2, influenza-associated pulmonary aspergillosis, Salmonella and Plasmodium burgdorferi, etc.), and DLBCL patients, the expression level of La is significantly positively correlated with CSR / SHM activity in physiological immune response, while it is positively correlated with immunoglobulin heavy chain-Myc translocation frequency and genomic instability in tumorigenesis, providing quantifiable biomarkers and bidirectional intervention targets for clinical practice.

[0051] 4) Technical solution is feasible: La can be expressed in B cells through genetic engineering methods (such as lentiviral vectors and Tet-On induction system), providing an operable implementation plan for immune reconstitution, vaccine adjuvants and antibody engineering. Attached Figure Description

[0052] Figure 1 The amino acid sequence alignment results of Lupus La protein (La) in human and mouse sources in this embodiment of the invention show that mouse La (SEQ ID NO:1) and human La (SEQ ID NO:2) have approximately 95% sequence identity.

[0053] Figure 2 This is a schematic diagram illustrating the mechanism by which Lupus La protein (La) acts as a structural bridge to mediate AID targeting the immunoglobulin heavy chain (IgH) locus in this embodiment of the invention. Figure 2 The diagrams in the left and right images show the formation of an "AID-La-IgH" ternary complex, where La binds to the IgH 3′RR regulatory region and recruits AID to the CSR center (left image) and SHM center (right image), respectively. Figure 2 The B-mode model is a working model in which the absence of La leads to the inability of AID to target the CSR center and SHM center, thereby causing a decrease in antibody library diversity.

[0054] Figure 3 This is a diagram showing the construction and detection results of the CH12F3 cell line in the La protein regulation of CSR and SHM function verification experiment in this embodiment of the invention. Figure 3 In Figure A, Western blotting validation of La knockout (KO) and rescue is performed. Figure 3 B represents the IgA class switching efficiency of each group as detected by flow cytometry; Figure 3 In the middle, bar C represents the differences in IgA levels among the above cell lines; Figure 3 D represents the detection of CH12F3 using 5′Sμ as a decoy probe in CSR-HTGTS. La - / - , La - / - + Flag-EV and La - / - In the +Flag-La cell line Igh Frequency distribution of AID target sites at gene loci; Figure 3 The bar chart in section E shows the broken connections of the above cell lines in the donor Sμ region and the recipient Sα region; Figure 3 The SHM-seq analysis in the middle section shows the mutation frequency of the variable region CDR3 in the above cell lines; Figure 3 The middle G is a violin plot showing the comparison of CDR3 region mutation frequencies in the above cell lines. For Figure 3 Data for C, E, and G are from three independent replicate experiments and are presented as mean ± standard deviation. P Values ​​via unpaired double tails tThe results were obtained through testing and calculation. *** represents a significance analysis result of P < 0.001, and **** represents a significance analysis result of P < 0.0001.

[0055] Figure 4 This is a diagram showing the construction and detection results of the primary mouse B cell in vitro stimulation model in the La protein regulation of CSR and SHM function verification experiment in this embodiment of the invention. Figure 4 A is La A schematic diagram of a conditional gene knockout strategy. Figure 4 The B cell type was validated by Western blotting in B cell-specific La conditional knockout mice. Figure 4 C represents the flow cytometry analysis showing wild-type and La flox / flox Cd19-Cre Percentage of Pro-B cells and Pre-B cells in bone marrow of mice. Figure 4 The middle D is a bar chart showing wild type and La flox / flox Cd19-Cre The proportions of Pro-B cells and Pre-B cell subsets in mouse bone marrow were obtained from 5 independent mice and are presented as mean ± standard deviation. Figure 4 The middle D is the image showing the wild type and La flox / flox Cd19-Cre The size of the mouse's spleen; the red line shows the scale. Figure 4 Flow cytometry analysis of F showed wild-type and La flox / flox Cd19-Cre IgG1 levels in mice. Figure 4 The middle G is a bar chart comparing wild type and La flox / flox Cd19-Cre Differences in IgG1 levels in mice. Data from 6 independent mice are presented as mean ± standard deviation. Figure 4 H in the middle represents the flow cytometry analysis showing wild type and La flox / flox Cd19-Cre IgG3 levels in mice. Figure 4 The middle bar chart (I) compares wild-type and... La flox / flox Cd19-Cre Differences in IgG3 levels in mice. Data from 5 independent mice are presented as mean ± standard deviation. Figure 4 The genomes of the J group were harvested 96 hours after stimulation with LPS combined with IL4. CSR-HTGTS analysis using 5′Sμ as a bait probe showed that wild-type and La flox / flox Cd19-Cre In mice, Igh Frequency distribution of AID target sites at gene loci. Figure 4 The middle K is a bar chart showing the wild type and La flox / flox Cd19-Cre Disruptions in the Sμ, Sγ1, and Sε regions in mice were analyzed using data from three independent mice and are presented as mean ± standard deviation. Figure 4 The genome of L was harvested 96 hours after stimulation with LPS combined with αIgD-dextran. CSR-HTGTS analysis using 5′Sμ as a bait probe showed that wild-type and La flox / flox Cd19-Cre In mice, Igh Frequency distribution of AID target sites at gene loci. Figure 4 The middle M is a bar chart showing the wild type and La flox / flox Cd19-Cre Disconnection status in the Sμ, Sγ3, Sγ2b and Sγ2a regions in mice. Data from four independent mice are presented as mean ± standard deviation. Figure 4 In the middle N, Rep-SHM-seq sequencing was performed using JH1-4 as a decoy probe. The violin diagram illustrates this. La -cKO mice relative to wild-type mice and La Overall V in -cKO mice H Regional mutation frequencies, where each data point represents the average relative mutation frequency of a mutation site across three biological replicates. Box plots show the median, interquartile range, and data distribution range; scatter plots show the original data distribution. P-values ​​are calculated using paired two-tailed methods. t The results were obtained through verification and calculation. For Figure 4 D, G, I, K and M, P Values ​​via unpaired double tails t The results were obtained through verification and calculation.

[0056] Figure 5 This is a graph showing the molecular biological detection results from the La-IgH locus regulatory region verification experiment in this embodiment of the invention. Figure 5 In Figure A, electrophoretic mobility shift analysis (EMSA) shows the direct binding of the La protein to the 3′RR sequence of the immunoglobulin heavy chain. Figure 5 In Figure B, the enrichment signal of La in the IgH 3′RR region was detected by chromatin immunoprecipitation (ChIP) combined with qPCR. There was no significant difference in ns. * indicates that the significance analysis result is P<0.05, and ** indicates that the significance analysis result is P<0.01.

[0057] Figure 6This is a graph showing the protein interaction detection results in the direct interaction verification experiment between La and AID in this embodiment of the invention, demonstrating the binding of exogenous GFP-La and AID by co-immunoprecipitation (Co-IP).

[0058] Figure 7 The image shows the ChIP-qPCR results from the validation experiment of AID targeting the IgH locus in La-dependent manner in this embodiment of the invention. It compares the enrichment signal of AID protein in the S region of the IgH locus between wild-type (WT) and La knockout (KO) cells, indicating that La protein knockout significantly reduces the efficiency of AID protein targeting the IgH locus. In the figures, ** represents a significance analysis result of P < 0.01, *** represents a significance analysis result of P < 0.001, and **** represents a significance analysis result of P < 0.0001.

[0059] Figure 8 This is a visualization of single-cell RNA-seq (scRNA-seq) data from the correlation analysis of La expression levels with CSR activity in multiple pathogen infections (H1N1, SARS-CoV-2, PCV2, EBV, IAPA, Salmonella, and Plasmodium) in this embodiment of the invention. Figure 8 In multiple immune response models, A represents B cell subsets with high and low CSR levels. La Comparison of expression levels. Figure 8 B represents high and low levels under different immune response conditions. La The relationship between the proportion of cells expressing the gene and the level of CSR. Figure 8 C represents high expression La and low expression La B cell population IGHG , IGHA , IGHG and IGHG Transcript fold change (Log2 transformation). Figure 8 D is highly expressed La and low expression La The correlation between DNA damage repair and AID targeting activity in B cell populations was studied. Data were obtained from eight independent datasets, and statistical analysis was performed using paired two-tailed methods. t test.

[0060] Figure 9 This is a graph showing the dynamic changes in La expression and CSR / SHM after SARS-CoV-2 mRNA vaccination in this embodiment of the invention. Figure 9 In the diagram, A and B represent the UMAP clustering of immune cells in the draining lymph nodes, respectively (A) and (B). La Gene expression feature map (B), with the B cell region of the germinal center marked by a dashed box. Figure 9 C is La The distribution ratio of CSR levels between low-expression and high-expression groups. Figure 9 D represents the dynamic changes in CSR levels in germinal center B cells at different days after inoculation. Figure 9 E in the figure represents the UMAP subgroup of B-cell subsets in the draining lymph nodes of vaccine recipients. Figure 9 The middle F-plot shows the high and low levels of CSR in B cells of germinal centers. La The differences in expression were statistically analyzed using the Wilcoxon rank-combination test. Figure 9 G is La Different antibody types expressed in high- and low-expression germinal center B cells ( IGHA , IGHG , IGHD and IGHM ) Composition ratio. Figure 9 H is La The distribution ratio of somatic SHM levels in B cells of germinal centers with high and low expression. Figure 9 In Figure I, B cells from the germinal center at different days after vaccination are represented. La Covariation dynamics of expression, SHM level, and CSR status. The outer circle represents... La Expression levels, with the middle circle representing SHM levels and the inner circle representing CSR status. The data represent the proportion of cells in each population.

[0061] Figure 10 This is a graph showing the correlation between the prognosis of SARS-CoV-2 infected patients and La expression and CSR levels in an embodiment of the present invention; Figure 10 Image A shows the UMAP clustering of B cells in PBMCs of COVID-19 patients, with the plasma cell population marked by a dashed box. Figure 10 In the B group, CSR levels were found in both low and high levels in plasma cells from COVID-19 patients. La For comparison of expression levels, the Wilcoxon rank-combination test was used for statistical analysis. Figure 10 C represents plasma cells in patients in the improvement group and the non-improvement group. La , IGHM and IGHG Comparison of expression levels. Figure 10 D represents the plasma cells in the improved group and the non-improved group. La Joint analysis of expression level and CSR level.

[0062] Figure 11 The image shows the results of high-throughput whole-genome translocation sequencing (HTGTS) in the verification experiment of increased genome instability caused by La overexpression in this embodiment of the invention. Figure 11 In Figure A, Western blot analysis was performed to verify the overexpression of La in the CH12F3 cell line. Figure 11Figure B shows the Circos diagram illustrating the genome-wide translocation distribution between the CH12F3 control group and the La overexpression group (using c-Myc as bait). Figure 11 The bar chart in the middle (C) shows the number of AID off-target hotspots and off-target junctions, indicating that LLCP overexpression significantly increases AID-mediated off-target activity (P<0.001).

[0063] Figure 12 This image shows the single-cell sequencing results of patients with diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and transformed follicular lymphoma (tFL) in the analysis of La expression and clinical significance in B-cell lymphoma in this embodiment of the invention. Figure 12 B cells in DLBCL, MCL, FL, and tFL lymphomas are... La Expressing horizontal distribution. Figure 12 C represents high and low expression in DLBCL, MCL, FL, and tFL lymphomas. La The proportional distribution of genes in cells. C. Distribution of CIN70 chromosomal instability characteristics in B cells of DLBCL, MCL, FL, and tFL lymphomas. Figure 12 In the middle D, the CIN70 score is represented by the number of DLBCL, MCL, FL, and tFL lymphoma B cells. La The correlation scatter plot of gene expression levels, correlation coefficients and significance levels were calculated using the Spearman rank correlation test. Figure 12 E represents DLBCL, MCL, FL, and tFL lymphoma B cells. c-MYC , IGHG and IGHA Gene expression levels.

[0064] Figure 13 This is a graph showing the whole genome sequencing (WGS) results of different B-cell lymphoma patients in the La and Igh-Myc translocation correlation analysis in this embodiment of the invention. Figure 13 In diagram A, a Circos plot shows the lymphomas of DLBCL, MCL, FL, and tFL in patients. c-MYC and IGH Structural variations and translocation patterns between constant regions within a gene locus; blue arcs represent chromosomal translocation connections, and numbers represent those detected in various types of lymphoma. c-MYC / IGH Number of chromosomal translocations. Figure 13 B represents different types of lymphoma patients. c-MYC Towards IGH A statistical chart showing the proportion of patients with translocations at gene loci (including V(D)J and constant regions). Figure 13 C represents different lymphoma patients. c-MYC Towards IGH A statistical chart showing the proportion of patients with 3′RR translocations within a gene locus. Detailed Implementation

[0065] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0067] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0068] In the following embodiments, the cell culture, gene editing, virus packaging, flow cytometry, sequencing analysis, and animal experiment methods all employ conventional experimental techniques in the art.

[0069] Example 1 The amino acid sequence of the mouse Lupus La protein (Mus musculus La, UniProt accession number P32067, full-length amino acid sequence, 415 aa) is shown in SEQ ID NO:1, and the amino acid sequence of the human Lupus La protein (Homo sapiens La, UniProt accession number P05455, full-length amino acid sequence, 408 aa) is shown in SEQ ID NO:2. Sequence alignment of the two proteins is shown in [link to SEQ ID NO:2]. Figure 1 .

[0070] The full-length amino acid sequence of the human Lupus La protein (Homo sapiens La) shares 95% sequence identity with the mouse Lupus La protein (Mus musculus La), and is expected to have the same biological function. Based on the high sequence and functional conservation between human La and mouse La, those skilled in the art can directly apply the functional data of the mouse La described in this invention to human La without additional inventive effort. The full-length amino acid sequence of the mouse Lupus La protein (Mus musculus La) is used in all functional verification experiments of this invention.

[0071] The murine Lupus La protein is encoded by the Ssb gene, which is located on mouse chromosome 3 (NCBI accession number: NC_000068.8, Gene ID: 20823) and contains 7 exons. SEQ ID NO: 3 shows the coding sequence (cDNA) of this gene, encoding a complete open reading frame of 415 amino acids.

[0072] The applicant, through in vitro and in vivo experiments, has for the first time discovered that the Lupus La protein (abbreviated as La) can act as a structural bridge, specifically binding to the regulatory region of the immunoglobulin heavy chain locus and directly recruiting AID to the CSR / SHM site, forming an "AID-La-immunoglobulin heavy chain" functional complex. This facilitates the precise targeting of AID in a transcriptional-independent manner. The mechanism by which La protein acts as a structural bridge mediates AID targeting the immunoglobulin heavy chain (IgH) locus is described in [link to relevant documentation]. Figure 2 . Figure 2 The diagrams in the left and right images show the formation of an "AID-La-IgH" ternary complex, where La binds to the IgH 3′RR regulatory region and recruits AID to the CSR center (left image) and SHM center (right image), respectively. Figure 2 The model in section B is a working model where La deletion prevents AID from targeting the CSR and SHM centers, leading to a decrease in antibody library diversity. The following experiments validate the mechanism by which La protein acts as a structural bridge mediating AID targeting the IgH locus: I. Construction of La expression vector and establishment of stable cell lines 1. Construction of La expression vector 1.1 Obtaining the cDNA sequence encoding the full-length La protein: In this experiment, the cDNA sequence (SEQ ID NO:3) encoding the full-length La protein (SEQ ID NO:1) was obtained using reverse transcription. Total RNA was extracted from the CH12F3 mouse B lymphoma cell line (described in the literature "High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells") as the biological material. Using 1 μg of total RNA as a template, reverse transcription was performed using the Novizumi second-generation full-length cDNA first-strand synthesis kit (Nanjing Novizumi Biotechnology Co., Ltd., catalog number: R211-01). Oligo(dT) primers were used for reverse transcription. 23 VN and Random hexamers were mixed in equal proportions to ensure consistent reverse transcription efficiency across all regions of the mRNA. The specific procedures were performed according to the kit instructions. The resulting cDNA was used as a PCR template to amplify the gene sequence encoding the full-length La protein.

[0073] 1.2 Construction of the Flag-La eukaryotic expression vector: The above cDNA sequence was cloned into the pSin-EF1α-3×FLAG eukaryotic expression vector (described in the literature "Wu, G. et al. Menin enhances c-Myc-mediated transcription to promote cancer progression. Nat. Commun. 8, 15278 (2017), obtainable from our laboratory"). The specific steps are as follows: Vector linearization: using restriction endonucleases Cla I (New England Biolabs product, catalog number: R0197V) and BamH I (NEB product, catalog number: R0136V) was used to double digest the pSin-EF1α-3×FLAG vector to recover the linearized vector backbone.

[0074] Insert fragment amplification and restriction enzyme digestion: Using cDNA obtained from reverse transcription as a template, the La protein coding sequence was amplified using the following specific primer pairs: F1: 5′GGGGATCGATTACAAGGATGACGATGACAAGgctgaaaatggagataatgaaaaaatg-3′ (where the bolded part is the ClaI restriction site ATCGAT); R1: 5′-GGGGGGGATCCctacttgtctctagcaccattttctc-3′ (where the bolded part is the BamHI restriction site GGATCC).

[0075] The PCR product was digested with ClaI and BamHI, and the target fragment (approximately 1200 bp) was recovered.

[0076] Ligation reaction: The double-digested insert fragment and the linearized vector were mixed at a molar ratio of 3:1 to 10:1, and T4 DNA ligase (NEB product, catalog number: M0202V) was added. The mixture was incubated at 16°C to catalyze the ligation of sticky ends, thus constructing the pSin-EF1α-Flag-La recombinant expression plasmid.

[0077] 1.3 Construction of the GFP-La eukaryotic expression vector: A fusion expression vector for EGFP and La protein was constructed using a homologous sequence-based in vitro recombination method (Gibson Assembly, also known as seamless cloning). The vector backbone was pSin-EF1α-3×FLAG (or other derived vectors with puromycin resistance selection markers), which was linearized by double digestion with restriction endonucleases EcoRI (NEB, catalog number: R3101V) and BamHI (NEB, catalog number: R3136V). The specific steps are as follows: EGFP fragment amplification: Using a plasmid containing the EGFP coding sequence (pEGFP-C1 in this example, a product of Newbio Biotechnology, catalog number: V012024) as a template, the EGFP coding sequence was amplified using the following primers: F2: 5′ccatttcaggtgtcgtgaggGAATTCgattatcgccATGGTGAGCAAGGGCGAG-3′ (where the bolded part is the EcoRI restriction site GAATTC). R2: 5′cattttcagcAGATCCTCCTCCTCCCTTGTACAGCTCGTCCATGC-3′ (where the underlined part is the homologous arm sequence complementary to the upstream primer of the La gene).

[0078] La fragment amplification: Using cDNA obtained from reverse transcription as a template, the La protein-coding sequence was amplified using the following primers: F3: 5′gctgtacaagGGAGGAGGAGGATCTGCTGAAAATGGAGATAATGAAAAAATG-3′ (where the bolded part is the homologous arm sequence complementary to the downstream primer of the EGFP fragment). R3: 5′-ggccgccctagatgcatgcGGATCCCTACTTGTCTCTAGCACC-3′ (where the bolded part is the BamHI restriction site GGATCC).

[0079] Homologous recombination ligation: The EcoRI / BamHI linearized vector backbone, EGFP amplified fragment, and La amplified fragment were mixed at a molar ratio of 1:2:2, and Gibson Assembly Master Mix (NEB product, catalog number: E2611S) was added. The mixture was incubated at 50°C for 15 to 60 minutes. Through a recombination reaction mediated by terminal homologous sequences, the EGFP and La genes were sequentially ligated into the vector to construct the pSin-EF1α-EGFP-La recombinant expression plasmid.

[0080] 2. Construction of stable cell lines (lentiviral system) Using viral packaging and transfection methods, the various recombinant expression vectors constructed above were introduced into B cells (such as CH12F3 cell line or primary mouse spleen B cells; CH12F3 cells were specifically used in this example). After antibiotic selection and Western blot verification, cell lines stably expressing or induced to express La were established. The specific steps are as follows. Lentiviral packaging: Low-passaged (P<10) 293T cells (ATCC product; catalog number: RL-11268) were seeded in 10 cm dishes, and confluence was confirmed to be 70%-80% the next day. 14 μg of total DNA was diluted in 1 mL of basal DMEM (Corning product; catalog number: 10-013-CVRC) at a recombinant expression vector ratio of pMD2G: psPAX2: La = 1:2:4. Separately, 100 μL of PEI (Sigma product; catalog number: 408727) (1 mg / mL) was added dropwise, and the mixture was incubated at room temperature for 10 min before transfecting 293T cells. The supernatant was collected after 48 h and filtered through a 0.45 μm PVDF filter to obtain the viral stock solution. It was used immediately or aliquoted and stored at -80°C.

[0081] Lentiviral infection and drug screening: Take 1×10⁻⁶ CH12F3 cells in logarithmic growth phase. 6 Cells were seeded per well in 12-well plates, and 1 mL of complete culture medium containing 8 μg / mL Polybrene (Sigma, catalog number: TR-1003) was added. An equal volume of virus stock solution was added, and the plates were centrifuged at 2,700 rpm for 1.5 h to infect the cells, followed by 24 h of further culture. Cells were collected 24 h post-infection and resuspended in complete culture medium containing 800 ng / mL Puromycin (APEXBIO, catalog number: B7587) in 6-well plates. An uninfected control group was included to monitor the effectiveness of the drug screening. Successful infection was defined as 100% cell death in the control group and a survival rate ≥30% in the experimental group after 48-72 h.

[0082] Protein level verification: Cells were collected on days 4-5 after drug screening (1×10⁻⁶). 6Cells / sample), lysed in 1×Laemmli buffer (containing 100 mMDTT), in a 100°C metal bath for 10 min, centrifuged at 12,000 rpm for 2 min, and the supernatant collected. Separated using 12% SDS-PAGE (flattened at 80V, then electrophoresed at 120V for 50-60 min), wet-transferred to a PVDF membrane (300 mA constant current, 4°C, 1.5 h). Blocked with 5% skim milk powder for 1 h, then incubated overnight at 4°C with anti-GFP antibody (Abcam, 184601, 1:1000). Incubated with HRP-goat anti-mouse secondary antibody (1:5,000) at room temperature for 1 h, and developed with ECL chemiluminescence reagent. Expected band approximately 75 kDa (La 47.6 kDa + GFP tag).

[0083] The cell line obtained by introducing the vector Flag-La (i.e., pSin-EF1α-Flag-La) into the CH12F3 cell line is called the Flag-La cell line.

[0084] The cell line obtained by introducing the vector GFP-La (pSin-EF1α-EGFP-La) into the CH12F3 cell line is called the GFP-La cell line.

[0085] II. Cellular-level validation of La-regulated CSR and SHM function Material: Cells: CH12F3 cells CRISPR plasmid: pX330-Cas9-CMV-mCherry (described in the literature "Cell Reports: RAG2 abolishes RAG1 aggregation to facilitate V(D)J recombination").

[0086] La Gene-targeting gRNA: 5'-GAACAGATCAAATTGGATGA-3' (SEQ ID No. 4); 5'-GATCAAATTGGATGAAGGCT-3' (SEQ ID No. 5); 5'-GATGATGATGATCGTCGTCG-3' (SEQ ID No. 6); 5'-GGTAATAGGCCTGGTTATGC-3' (SEQ ID No. 7).

[0087] La Construction and validation of gene knockout and complement cell lines: Constructed using CRISPR-Cas9 technology La The gene knockout (KO) cell line, specifically the CH12F3 cell line edited using CRISPR-Cas9 technology, was constructed using the following knockout vector: [The text then abruptly shifts to a seemingly unrelated topic about gene knockout vectors.] La The four gRNAs of the gene were cloned pairwise into pX330-Cas9-CMV-mCherry to obtain the knockout vector pX332-gRNA-Cas9 (expressing both gRNA and Cas9). This vector was then transfected into CH12F3 cells, and single clones were screened using the limiting dilution method. Western blotting was used to verify the La protein knockout efficiency. La Gene knockout (KO) cell lines are called La - / - .

[0088] Through viral transfection methods La Gene knockout ( La - / - The exogenous La was reintroduced into the cell line. The specific viral transfection method is described in section 2 of Part 1 of this embodiment. The vector expressing La was the Flag-La (i.e., pSin-EF1α-Flag-La) constructed in section 1.2 of this embodiment. The resulting cell line after reintroduction was... La - / - +Flag-La.

[0089] In the same way La Gene knockout cells ( La - / - Obtained by transferring the Flag-EV (i.e., pSin-EF1α-3×FLAG) vector into the medium La - / - +Flag-EV is the control cell line.

[0090] Regarding the acquisition La Gene knockout cell lines La - / - and the replenished cell line La - / - +Flag-La was used for Western blotting validation, and the results are shown below. Figure 3 In the middle A, the first cell from the left is CH12F3, and the second cell from the left is the knockout cell line. La - / - The two strains on the far right are the replenished cell lines numbered #1 and #2, respectively. La - / - +Flag-La (Confirmation), third from the right is the control cell line. La - / - +Flag-EV. Western blot validation showed that the knockout and replacement cell lines were successfully constructed.

[0091] In vitro stimulation of cell lines to induce CSR: Targeting wild-type (WT) CH12F3 and knockout cell lines La - / - Replenishment of cell lines La - / - +Flag-La and control cell lines La - / - +Flag-EV uses in vitro stimulation (anti-CD40 / IL-4 / TGF-β or LPS / IL-4, etc.) to induce CSR.

[0092] The specific stimulating factors (cell lines) are: αCD40 (eBioscience product, 14-0401-81), IL-4 (PeproTech product, 200-04), and TGF-β (R&D Systems product, 240-B-010).

[0093] The above cell lines were placed in 24-well plates (2×10⁶ cells / wells). 5 Cell lines were stimulated for 72 h with αCD40 (1 μg / ml), IL-4 (20 ng / ml), and TGF-β (0.5 ng / ml), respectively, per well. Three replicates were set up.

[0094] Flow cytometry analysis of conversion efficiency: Cells were collected after stimulation, stained with PE-anti-IgA antibody (eBioscience product; catalog number: 12-4204-83), and the IgA / IgG conversion rate was detected by flow cytometry.

[0095] Flow cytometry antibodies used: PE anti-mouse IgA (eBioscience, Cat#12-4204-82, RRID: AB_465917), FITC anti-mouse IgG1 (BD Biosciences, Cat#553443), and FITC anti-mouse IgG3 (BD ​​Biosciences, Cat#553403).

[0096] See results Figure 3 B indicates the La KO group cell line. La - / - The IgA conversion rate dropped to single digits (<10%), which was significantly lower than that of the control group CH12F3 (approximately 50%).

[0097] High-throughput sequencing (CSR-HTGTS) was used to detect the efficiency of DNA double-strand breaks ligation and recombination induced by AID targeting. Wild-type (WT) cell line CH12F3 and knockout cell line were extracted separately after the above stimulation. La - / - Genomic DNA was analyzed, and the efficiency of DNA double-strand breaks ligation and recombination caused by AID targeting each S region of the immunoglobulin heavy chain was detected using the Sμ region as bait, in accordance with the HTGTS standard procedure (PMID: 26308889).

[0098] See results Figure 3 CSR-HTGTS display La - / - The targeting of AID in the Sμ and Sα regions resulted in a significant decrease in the efficiency of DNA double-strand breaks and recombination.

[0099] V(D)J region somatic high-frequency mutation (SHM) frequency: extracted from wild-type (WT) cell line CH12F3 and knockout cell line after stimulation. La - / - Genomic DNA was used to amplify the CDR3 region by PCR, and mutation frequency was analyzed by high-throughput sequencing.

[0100] See results Figure 3 D in the middle indicates La - / - The efficiency of SHM in the CDR3 region is significantly reduced.

[0101] III. Validation of La in vivo and in vitro function in mouse models Constructing a B cell-specific La conditional knockout (Cd19-Cre; La-flox) mouse model: Material: Mouse: Cd19-Cre (C57BL / 6 background) The animal research program involved in this application was approved by the Experimental Animal Welfare and Ethics Committee of Peking University, and the research operations were conducted in accordance with the 8th edition of the "Laboratory Animal Care and Use Manual". This invention uses two types of mice: those purchased from Jicui Pharmaceutical Co., Ltd. La Conditional knockout mice (Strain ID: T018910) and mice purchased from Cyagen Biosciences Co., Ltd. Cd19-Cre The tool mouse (Strain ID: C001479), by crossing two types of mice, can obtain B-cell specific... La Conditional knockout mice. This hybridization strategy is based on the Cre-LoxP specific recombination system, in which... Cd19 Promoter-driven Cre recombinases can specifically recognize and cleave early in B cell development. La The loxP sites flanking the gene are aligned in the same direction, thus achieving [the desired effect] in B-cell lineages. LaThe genes are precisely identified without affecting other tissues and organs. La Normal physiological functions of proteins.

[0102] Conditional knockout mice were designed and constructed based on the C57BL / 6 mouse background, according to... La The gene sequence was used to insert two identical LoxP elements upstream of exon 3 and downstream of exon 6, respectively. Figure 4 (A). In the presence of Cre recombinase, exons 3 through 6 between the two LoxP sites will be specifically cleaved, thereby achieving... La Functional knockout of genes. To obtain a B-cell-specific knockout model, this invention combines it with... Cd19-Cre The tool mice were hybridized. Cd19 Promoter-driven Cre recombinases can begin to be expressed early in B cell development, ensuring... La The gene was efficiently and specifically deleted in the B-cell lineage, while preserving normal La expression in other immune cells and tissues. By extracting DNA from the toe paws of progeny mice for PCR genotyping, this invention successfully screened and obtained wild-type mice (hereinafter referred to as Wide-type) and B-cell-specific La conditional knockout mice with consistent genetic backgrounds. La flox / flox Cd19-Cre ). Then collect La Splenic B cells from conditional knockout mice and their littermate wild-type mice were analyzed using immunoblotting to detect La protein expression levels. The results showed that, compared to wild-type mice, La flox / flox Cd19-Cre The expression level of La protein in mouse spleen B cells was significantly decreased, but a weak residual band could still be detected. Figure 4 (B)

[0103] Early B cell isolation from bone marrow: Mice were euthanized by cervical dislocation, and the bilateral femurs and tibias were aseptically isolated and placed in 2.5% FBS / PBS solution. The bone tissue was ground with a 10 mL syringe tip until the bone marrow cavity turned white, filtered through a 70 μm cell sieve into a 15 mL centrifuge tube, and centrifuged at 2200 rpm for 5 minutes at 4°C, discarding the supernatant. The pellet was resuspended in 5 mL of ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, and 0.01 mM Na2EDTA) and incubated at room temperature for 3 minutes to lyse red blood cells. Lysis was then terminated by adding 10 mL of 2.5% FBS / PBS, and centrifuged at 2200 rpm for 5 minutes at 4°C, discarding the supernatant. The pellet was resuspended in an appropriate amount of 2.5% FBS / PBS, and CD43-FITC, IgM-APC, and B220-APC-Cy7 antibodies were added at a 1:200 ratio, incubating at room temperature in the dark for 15 minutes. Wash with 2 mL of 2.5% FBS / PBS, centrifuge at 2200 rpm for 5 minutes at 4°C, discard the supernatant, resuspend the pellet in 2.5% FBS / PBS, and sort using a BD FACSAria III flow cytometer. Sorting strategy: Pro-B cells were sorted using B220. + IgM - CD43 + Pre-B cells are B220 + IgM - CD43 - Genomic DNA was extracted from the sorted cells using the QIAampDNA Mini Kit (QIAGEN, 51306).

[0104] Given that abnormal early B cell development may indirectly affect subsequent CSR and SHM processes, this invention first focuses on wild-type and... La flox / flox Cd19-Cre Bone marrow development and stimulated lymphoid organ structure in mice were examined to ensure the reliability of subsequent phenotypic analysis. This invention first isolated wild-type and... La flox / flox Cd19-Cre Mouse bone marrow cells were quantitatively analyzed using flow cytometry to detect key stages of early B cell development, and Pro-B cells (B220) were detected. + CD43 + IgM - ) cells and Pre-B cells (B220) + CD43 - IgM - The results showed that the ratio of wild type to... La flox / flox Cd19-CreThe proportions of mice at the above developmental stages did not differ significantly. Figure 4 (C, D) This indicates that La protein deficiency does not affect the early differentiation, proliferation, and maturation of B cells in the bone marrow. Furthermore, to assess the impact of La protein deficiency on the overall development of secondary lymphoid organs, this invention performed gross morphological measurements on the mouse spleen. The results showed that... La flox / flox Cd19-Cre The size of the spleen in mice was not significantly different from that in the wild-type control group. Figure 4 The results (E) indicate that the absence of La protein has no significant effect on spleen development and structural integrity.

[0105] Isolation and in vitro stimulation of mouse spleen B cells: Stimulating factors (mouse spleen B cells): LPS (PeproTech product, 500-P257), IL-4 (PeproTech product, 200-04), and LPS (25 μg / ml, PeproTech product, 500-P257) and αIgD-dextran (Fina BioSolutions product, FinaBio0001).

[0106] Splenic B cells were isolated and subjected to in vitro stimulation (LPS / IL-4 co-stimulation or LPS / anti-IgD-dextran co-stimulation) to induce class switching; La-flox conditional knockout mice were sacrificed. La f / f Cd19-Cre Spleens were obtained from littermates and wild-type controls. Single-cell suspensions were prepared by grinding. CD43-positive cells were removed using the Mouse B Cell Isolation Kit (Vazyme, CS201-01), and the negative fraction was obtained as resting B cells (purity >95%). The B cells were then placed in 24-well plates (0.5 × 10⁻⁶). 6 Cells were divided into two groups of stimuli per well: ① LPS (25 μg / ml) + IL-4 (20 ng / ml); ② αIgD-dextran (3 ng / ml). Cells were collected after 96 h of culture and at least three biological replicates were set up.

[0107] Flow cytometry detection of IgG1 or IgG3 conversion efficiency: The collected stimulated cells were stained with FITC-anti-IgG1 (BD Biosciences, catalog number: 553443) or FITC-anti-IgG3 antibody (BD Biosciences, catalog number: 553403), and the IgG1 or IgG3 conversion efficiency was detected by flow cytometry.

[0108] See results Figure 4 Under the stimulation of F, G, LPS+IL-4, La Gene knockout group La flox / flox Cd19-Cre The IgG1 conversion rate decreased from 35% at WT to 1.5%; under αIgD-dextran stimulation, La flox / flox Cd19-Cre The IgG3 conversion rate decreased from 10% in WT to 1%.

[0109] High-throughput sequencing was used to detect the efficiency of DNA double-strand breaks ligation and recombination caused by AID targeting the S region. Genomic DNA was extracted from the cells and, following the HTGTS standard procedure (PMID: 26308889), using the Sμ region as bait, the targeting enrichment signal of AID in each S region of the immunoglobulin heavy chain was detected.

[0110] See results Figure 4 JM, CSR-HTGTS showed two stimulation conditions, La KO mice La flox / flox Cd19- Cre The DNA double-strand breaks caused by AID targeting each S region result in a decrease in recombination efficiency to undetectable levels.

[0111] Isolation of Peyer's patch germinal center B cells: Peyer's patch was harvested along the mesenteric side of the small intestine, ground, filtered through a 70 μm screen, and centrifuged at 2200 rpm for 5 min to remove the supernatant. PNA-positive GC B cells (GL7+Fas+) were enriched according to the instructions of the Germinal Center B Cell (PNA) MicroBead Kit (Miltenyi, 130-110-479). Genomic DNA was extracted and Rep-SHM-seq was performed to obtain 88 VH regions, and high-throughput sequencing was used to analyze mutation frequencies.

[0112] See results Figure 4 N, La Gene knockout mice La flox / flox Cd19-Cre The SHM efficiency of all 88 VH regions in GC B cells decreased significantly.

[0113] All of the above results indicate that La deletion prevents AID from effectively targeting the immunoglobulin heavy chain locus, leading to CSR failure and SHM reduction, resulting in a decrease in antibody library diversity (Repertoire Collapse).

[0114] IV. Verification of La binding to immunoglobulin heavy chain loci To verify the interaction between the La protein and the 3′RR enhancer, biotinylated probes covering the four core HS sequences (HS3a, HS1, HS2, HS3b, HS4) were designed and synthesized. EMSA experiments using purified His-La protein showed that the La protein specifically binds to each core sequence, forming protein-DNA complex bands. Figure 5 (A), confirming the direct binding of the two in vitro. Further ChIP-qPCR experiments using Flag-La stable cell lines revealed that La protein was significantly enriched in the four core element regions, while no significant signal was observed in the 3′RR internal spacer sequence and the Gapdh site. Figure 5 (B) indicates that under physiological conditions, the La protein can also specifically bind to the 3′RR enhancer.

[0115] Chromatin immunoprecipitation (ChIP) combined with qPCR was used to detect the enrichment signal of La protein at the immunoglobulin heavy chain locus; electrophoretic mobility shift analysis (EMSA) was used to verify the direct binding ability and sequence specificity of La protein to the 3′RR regulatory region of immunoglobulin heavy chain.

[0116] V. Verification of the interaction between La and AID This invention utilizes a previously constructed cell line stably expressing the GFP-La fusion protein, employing GFP-Trap magnetic beads for affinity capture. This method, based on the specific recognition of GFP tags by nanobodies, effectively avoids non-specific interference from heavy and light chains during traditional antibody immunoprecipitation. Results are shown below. Figure 6 GFP-La can interact with endogenous AID proteins.

[0117] VI. Validation of AID targeting the immunoglobulin heavy chain locus in dependence on La To further verify that the La protein can directly regulate AID targeting Igh In this invention, ChIP-qPCR was further used to detect the enrichment levels of AID in the donor Sμ and receptor Sα regions of wild-type and La protein-deficient cells, respectively. The results showed that, compared with wild-type cells, the enrichment levels of AID in both the donor Sμ and receptor Sα regions were significantly decreased in La protein-deficient cells. Figure 7 This result indicates that the La protein is an effective target of AID. Igh Necessary conditions for locus rearrangement centers.

[0118] ChIP-qPCR experimental method: 1. Cell cross-linking and lysis CH12F3 cells were collected 36 hours after stimulation with anti-CD40-IL-4-TGFβ. 1 × 10⁶ cells were collected. 7 Cells were centrifuged and resuspended in PBS containing 1% formaldehyde. Incubation was performed at room temperature for 10 minutes to cross-link proteins and DNA. 2 M glycine was added to a final concentration of 125 mM, and incubation was performed at room temperature for 5 minutes to terminate the cross-linking. The cell pellet was washed twice with PBS and then resuspended in 1 mL of lysis buffer (5 mM HEPES, 85 mM KCl, and 0.5% NP40, containing 1× protease inhibitor). Lysis was performed on ice for 10 minutes, gently shaking every 5 minutes. After lysis, the pellet was collected by centrifugation and resuspended in 130 μL of nuclear lysis buffer (50 mM Tris-HCl, 10 mM EDTA, and 1% SDS, containing 1× protease inhibitor). Chromatin fragmentation was performed using a Covaris M220 focused sonication system with the following parameters: temperature 4 ℃, peak power 50.0, duty factor 40, Cycles / Burst 200, and processing time 6 minutes. DNA fragment sizes after sonication were controlled to be between 200 bp and 700 bp.

[0119] 2. Immunoprecipitation Transfer 130 μL of chromatin sample to a 1.5 mL centrifuge tube, add 1.3 mL of dilution buffer (16.7 mM Tris-HCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, and 167 mM NaCl, containing 1 × protease inhibitor), mix thoroughly, and centrifuge at high speed for 10 minutes. Collect the supernatant. Reserve 1 / 10 volume as an input control. Add the corresponding antibody to the remaining sample: for La protein detection, add 20 μL of ANTI-FLAG M2 magnetic beads; for AID protein detection, add 5 μg of AID antibody. Incubate overnight at 4°C. The next day, add 30 μL of Protein A magnetic beads to the AID group and continue incubation for 2 hours; no additional magnetic beads are needed for the FLAG group.

[0120] 3. Washing and elution The magnetic beads used to enrich the sample were washed once each with 800 μL of the following pre-cooled buffer: RIPA-LS (10 mM Tris-HCl, 140 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate and 1% Triton X-100, containing 1 × protease inhibitor). RIPA-HS (10 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate and 1% Triton X-100, containing 1 × protease inhibitor). RIPA-LiCl (10 mM Tris-HCl, 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL and 0.5% Na-Deoxycholate, containing 1 × protease inhibitor). 10 mM Tris-HCl, pH 8.0.

[0121] Discard the residual liquid, add 120 μL of elution buffer (1% SDS and 0.1 M NaHCO3), treat in a 65°C metal bath for 15 minutes, and collect the supernatant by instant centrifugation.

[0122] 4. Decross-linking and DNA purification Add NaCl to the elution buffer and input sample to a final concentration of 300 mM, then add 2 μL of proteinase K (20 mg / mL) and incubate at 65°C for 2 hours. After the reaction is complete, purify the DNA using the Zymo-Spin™ Column (D4014, Zymo) kit, and finally elute with 100 μL of nuclease-free water. Store at 20℃.

[0123] 5. qPCR detection qPCR reactions were performed using the Novizan Taq Pro Universal SYBR qPCR Master Mix kit. The total reaction volume was 10 μL, containing 5 μL of 2 × Master Mix, 0.2 μL each of 10 μM forward and reverse primers (see Table 1), and 4.6 μL of cDNA. The thermal cycling conditions were: 95 ℃ pre-denaturation for 30 seconds; 95 ℃ denaturation for 10 seconds, 60 ℃ annealing and extension for 30 seconds, for 40 cycles; melting curves were collected after the reaction. The detection instrument was a q225 real-time PCR instrument.

[0124] Table 1. Primer sequences for ChIP-qPCR detection: (5′–3′)

[0125] In wild-type (WT) and La knockout (KO) cells La - / -In this study, ChIP-qPCR was used to detect the enrichment signal of AID protein in the S region of the immunoglobulin heavy chain locus; the effect of La deletion on the spatial targeting efficiency of AID was compared and analyzed to verify the function of La as a structural bridge mediating AID targeting.

[0126] See results Figure 7 The study compared the enrichment signal of AID protein in the S region of the immunoglobulin heavy chain locus in wild-type (WT) and La knockout (KO) cells, indicating that knockout of La protein significantly reduced the efficiency of AID protein targeting the immunoglobulin heavy chain locus.

[0127] VII. Bioinformatics Analysis of La Expression and CSR Activity in Multiple Pathogen Infections Based on Public Datasets Download published single-cell RNA-seq datasets of B cells from multiple pathogen-infected patient / mouse models from public databases, including: Plasmodium berghei ANKA (CNGBdb: OMIX001839), influenza-associated pulmonary aspergillosis (IAPA, GEO: GSE290161), porcine circovirus type 2 (PCV2, GEO: GSE287481), influenza A virus subtype H1N1 (H1N1 subtype, GEO: GSE266469), systemic cytokine stimulation (Immune Dictionary, GEO: GSE202186), Epstein-Barr virus (EBV, GEO: GSE189141), SARS-CoV-2 (GEO: GSE176269), and Salmonella typhi (GEO: GSE122083) infection datasets. We used the Seurat package (v4.0+) for data quality control, standardization, and dimensionality reduction analysis to extract B cell subsets, calculate the expression level of the La gene, and perform correlation analysis (Spearman correlation test) with the expression levels of CSR-related genes (AICDA, IGHG, IGHA, etc.).

[0128] See results Figure 8 This indicates that, through CSR-related gene set module scoring and differential expression analysis of B cell subsets, it was found that B cells with high CSR levels in various pathogen infection models all exhibited high La expression, indicating that La expression level is significantly positively correlated with CSR activity. Figure 8 (A, B). Further comparison by median La expression revealed that in B cells with high La expression, the levels of IGHG and IGHA transcripts were significantly increased, while IGHM and IGHD were significantly decreased. Figure 8The characteristic of La (C) is consistent with the CSR conversion direction from IgM to IgG / IgA, suggesting that La may positively regulate CSR by promoting AID targeting efficiency. Furthermore, analysis of the module scores of the AID-related gene set (35 genes) and the DNA damage repair pathway (630 genes) revealed that the correlation coefficient between the two was significantly higher in the La high-expression population than in the low-expression population. Figure 8 The results (D) suggest that high La expression is synergistically positively correlated with AID recruitment and downstream DNA damage repair response.

[0129] VIII. Analysis of La expression and dynamic changes in CSR / SHM after SARS-CoV-2 mRNA vaccination To verify the regulatory role of La in CSR and SHM during physiological immune responses, this study integrated single-cell sequencing data from lymph nodes of SARS-CoV-2 mRNA vaccine recipients (Kim et al., scRNA-seq+scBCR-seq; Dhenni et al., scRNA-seq). The vaccination model induces a strong germinal center response, and the draining lymph nodes provide an ideal system for studying the function of La in CSR.

[0130] Based on data from Kim et al., UMAP clustering showed that germinal center B cells exhibited significant La high expression signals ( Figure 9 (A, B). Germinal center B cells were grouped according to the median La value. The proportion of cells with high CSR levels was significantly increased in the La-high expression population. Figure 9 The proportion of CSR cells increased with inoculation time, while the proportion of low-level CSR cells decreased. Figure 9 (D) suggests that La may play a gradual regulatory role in the dynamic maturation of germinal centers.

[0131] The above results were validated in independent datasets by Dhenni et al., showing that the expression level of La in the high-CSR population of germinal center B cells was significantly higher than that in the low-CSR population. Figure 9 (E, F).

[0132] Further analysis of scBCR-seq data revealed that La-overexpressing germinal center B cells showed increased proportions of IGHG and IGHA, and decreased proportions of IGHD and IGHM. Figure 9 (G), consistent with the direction of CSR; at the same time, its proportion of high-level somatic high-frequency mutant (SHM) cells is significantly higher ( Figure 9 (H). Time-series analysis showed that the proportion of cells with high La expression, high SHM levels, and completed CSR increased synchronously from day 35 to day 110. Figure 9 (I) indicates that La continuously promotes AID-mediated CSR and SHM processes in vaccine-induced germinal center responses.

[0133] IX. Correlation analysis between prognosis and La expression in SARS-CoV-2 infected patients Peripheral blood single-cell sequencing data of COVID-19 patients (E-MTAB: 9357) were downloaded from public databases. Plasma cell / B cell subsets were extracted from recovered and non-improved patients, and the correlation between the expression levels of La, IGHM, and IGHG and CSR levels and disease prognosis was analyzed.

[0134] See results Figure 10 Patients were divided into an improvement group (recovery period) and a no-improvement group based on their clinical outcomes. Bubble charts showed that in the improvement group, La expression and IGHG levels in plasma cells were significantly increased, while IGHM levels were decreased; the no-improvement group showed the opposite pattern. Figure 10 (A) suggests that the recovery period involves an antibody class shift from IGHM to IGHG. Further combined stratified analysis showed that the proportion of cells with high CSR levels and high La expression was significantly higher in the improved group than in the non-improved group ( Figure 10 (B and C); combined distribution characteristics showed that high La expression and high CSR levels were synergistically increased in the improved group, while low La expression was associated with low CSR levels and unimproved clinical phenotypes ( Figure 10 (D). The above results suggest that high expression of La may promote immune clearance and clinical recovery of SARS-CoV-2 by enhancing CSR-mediated antibody response.

[0135] 10. Verification that La overexpression leads to increased genomic instability Cells and vectors: Cell line: CH12F3.

[0136] Vector: pSin-EF1α-EGFP-La (constructed in Part 1.3 of this example).

[0137] Control: Empty vector pSin-EF1α-GFP (the control vector was constructed by introducing the EGFP coding sequence into the vector backbone pSin-EF1α-3×FLAG, following the method described in Part 1.3 of this embodiment).

[0138] method: 1. Construction of stable cell lines (lentiviral system) Lentiviral packaging: Low-passaged (P<10) 293T cells (ATCC product; catalog number: RL-11268) were seeded in 10 cm dishes, and confluence was confirmed to be 70%-80% the next day. 14 μg of total DNA was diluted in 1 mL of basal DMEM (ATCC product, CRL-11268) at a molar ratio of pMD2G: psPAX2: pSin-EF1α-EGFP-La = 1:2:4. Separately, 100 μL of PEI (Sigma product; catalog number: 408727) (1 mg / mL) was added dropwise, and the mixture was incubated at room temperature for 10 min before transfecting 293T cells. After 48 h, the supernatant was collected and filtered through a 0.45 μm PVDF filter to obtain the viral stock solution. It was used immediately or aliquoted and stored at -80°C.

[0139] Lentiviral infection and drug screening: Take 1×10⁻⁶ CH12F3 cells in logarithmic growth phase. 6 Cells were seeded per well in 12-well plates, and 1 mL of complete medium containing 8 μg / mL Polybrene was added. An equal volume of virus stock solution was added, and the plates were centrifuged at 2,700 rpm for 1.5 h to infect the cells, followed by 24 h of further culture. Cells were collected 24 h post-infection and resuspended in complete medium containing 800 ng / mL Puromycin in 6-well plates. An uninfected control group was included to monitor the effectiveness of the drug screening. Successful infection was defined as 100% cell death in the control group and a survival rate ≥30% in the experimental group after 48-72 h.

[0140] Protein level verification: Cells were collected on days 4-5 after drug screening (1×10⁻⁶). 6 Cells / samples were lysed in 1×Laemmli buffer (containing 100 mMDTT), bathed at 100℃ for 10 min, and centrifuged at 12,000 rpm for 2 min to collect the supernatant. Separation was performed using 12% SDS-PAGE (flattened at 80V, electrophoresis at 120V for 50-60 min), followed by wet transfer to a PVDF membrane (300 mA constant current, 4℃, 1.5 h). After blocking with 5% skim milk powder for 1 h, the cells were incubated overnight at 4℃ with anti-GFP antibody (Abcam, 184601, 1:1,000). Incubation with HRP-goat anti-mouse secondary antibody (1:5,000) at room temperature for 1 h was followed by ECL luminescence. The expected band was approximately 75 kDa (La 47.6 kDa + GFP tag). The two cell lines overexpressing La were named GFP-La#1 and GFP-La#2, respectively.

[0141] Following the method described in Part II of this embodiment, La was knocked out of the CH12F3 cell line via viral transfection, resulting in two La-knockout cell lines, namely... La- / - #1 and La - / - #2.

[0142] Two cell lines overexpressing La (GFP-La#1 and GFP-La#2) and two cell lines knocking out La ( La - / - #1 and La - / - #2) and the CH12F3 cell line, the results of Western Blot validation are shown in […]. Figure 11 In cell line A, the overexpression of La in GFP-La#1 and GFP-La#2 was verified.

[0143] 2. CSR-HTGTS detection of off-target translocation High-throughput whole-genome translocation sequencing (HTGTS) was used with c-Myc as a decoy to detect the frequency of AID-mediated off-target translocation across the entire genome in the CH12F3 cell line and La-overexpressing cell lines. Genomic DNA was extracted from the cells described above, and the AID genome-wide targeting enrichment signal was detected using the Sμ region as bait, following the HTGTS standard procedure (PMID: 26308889). Three replicates were set up.

[0144] To assess whether La overexpression induces genomic instability, this study constructed a CH12F3 cell line with stable La overexpression. Immunoblotting confirmed significantly high expression of exogenous La. Figure 11 (A). Subsequently, CSR-HTGTS sequencing was used to analyze AID-mediated genome-wide off-target translocations. The results showed that, compared to the control, the number of genome-wide off-target recombination connections was significantly increased in La-overexpressing cells (A). Figure 11 (B and C) indicates that La upregulation can induce abnormal DNA double-strand breaks at non-Ig loci of AID, thereby increasing abnormal connections with Myc loci and promoting chromosomal translocation events.

[0145] XI. Analysis of La expression and clinical significance in B-cell lymphoma based on public datasets Single-cell sequencing data of published B-cell lymphoma patients were downloaded from public databases, including: diffuse large B-cell lymphoma (DLBCL, GEO: GSE182434), follicular lymphoma and transformed follicular lymphoma (FL / tFL, https: / / doi.org / 10.11588 / DATA / VRJUNV; https: / / doi.org / 10.5281 / zenodo.6536723), mantle cell lymphoma (MCL, GEO: GSE303064), and DLBCL bulk RNA-seq data (GEO: GSE98588). B-cell subsets were extracted, and the correlation between La expression levels and genomic instability score (CIN70), MYC proto-oncogene, and IGHG / IGHA (CSR marker) expression levels was analyzed.

[0146] To investigate the expression of La in different lymphomas and its relationship with genomic instability, this study integrated scRNA-seq data from four types of lymphoma (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma (tFL), and mantle cell lymphoma (MCL)). The results showed that the expression level of La and the proportion of cells with high La expression were significantly higher in diffuse large B-cell lymphoma (DLBCL) than in other types. Figure 12 (A, B) indicates that La is collectively upregulated in DLBCL. As the most aggressive subtype, DLBCL also has a significantly higher CIN70 score than other subtypes ( Figure 12 (C), and La expression was significantly positively correlated with CIN70 score in all four types of lymphoma ( Figure 12 The expression of La (La) suggests that upregulation may be a common factor driving genomic instability in B-cell tumors. Further analysis showed that the expression of IgG / IgA and the off-target site MYC of AID were significantly increased in DLBCL. Figure 12 The results suggest that La may drive the accumulation of genomic instability by enhancing the genome-wide off-target activity of AID, thereby promoting CSR while mistargeting oncogenes such as MYC.

[0147] 12. Correlation Analysis of La and Igh-Myc Translocations Based on Public WGS Dataset Whole-genome sequencing (WGS) data of B-cell lymphoma patients obtained from published literature (PMID:38701426, PMID: 32584970, PMID: 30348671) were extracted to identify translocation breakpoints, structural variations, and genomic instability features in the Myc and Igh constant regions (S region and 3′RR region), and their correlation with La expression levels was analyzed.

[0148] Based on scRNA-seq data, samples from patients with four major types of B-cell non-Hodgkin lymphoma: DLBCL, FL, tFL, and MCL were analyzed. c-MYC A systematic comparative analysis of gene translocation characteristics was performed. The results showed that, compared with other types of lymphoma, DLBCL patients... c-MYC Towards IGH The frequency of constant regions within the locus was significantly higher than in other types of lymphoma, and translocation breakpoints were widely distributed in multiple S regions and 3′RR enhancer regions. Figure 13 (A). Meanwhile, events occur in DLBCL. c-MYC Towards IGH The proportion of patients with locus translocations (including constant and variable regions) is significantly higher than that of other types of lymphoma. Figure 13 (B), and occurred c-MYC The highest proportion of patients were directed to the 3′RR enhancing subregion. Figure 13 (C). In summary, the analysis results based on whole-genome sequencing data in this section, supplementing the transcriptome data mentioned above, provide supporting evidence for understanding the pro-cancer role of La protein in the development and progression of B-cell lymphoma from the perspective of genomic structural variations.

[0149] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application, characterized in that, The application refers to the use of Lupus La protein or related biomaterials of Lupus La protein in D1 or D2: The application of D1, Lupus La protein or related genetic material in improving the efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci, or in the preparation of drugs that improve the efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci. The genetic material is at least one of the following: c1. The nucleic acid molecule encoding the Lupus La protein; c2, expression cassettes, recombinant vectors, recombinant microorganisms, transgenic cell lines, transgenic animal tissues or transgenic organs containing the nucleic acid molecules described in c1; D2. The use of substances that knock out the Lupus La protein-coding gene or substances that target the Lupus La protein-coding gene in reducing the efficiency of activation-induced cytidine deaminase targeting the immunoglobulin heavy chain locus, or in the preparation of drugs that reduce the efficiency of activation-induced cytidine deaminase targeting the immunoglobulin heavy chain locus.

2. The application according to claim 1, characterized in that, The Lupus La protein is a protein of type A1, A2, or A3 as follows: A1. The amino acid sequence is the protein that is the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 in the sequence listing; A2. A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 in the sequence listing, which has more than 90% sequence identity with the protein shown in A1 and has the characteristics of A2.1-A2.4; A2.

1. It has a regulatory region that binds to immunoglobulin heavy chain loci; A2.2, Directly interacts with activation-induced cytidine deaminase; A2.3, Spatial targeting of activated cytidine deaminases in the transition or variable regions of immunoglobulin heavy chain loci; A2.

4. It has the function of promoting antibody class switching rearrangement and high-frequency mutation in somatic cells; A3, a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1 or A2.

3. The application according to claim 2, characterized in that, c1 The coding sequence of the nucleic acid molecule described is the nucleic acid molecule with SEQ ID No. 3 in the sequence listing.

4. The application according to any one of claims 1-3, characterized in that, The efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci is the antibody type switching rearrangement ratio and / or the somatic high-frequency mutation frequency.

5. The application according to claim 4, characterized in that, The drug described in D1 that enhances the efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci is either a drug that enhances vaccine response or a drug used to treat diseases caused by antibody type switching rearrangement defects.

6. The application according to claim 4, characterized in that, The substance used to knock out the Lupus La protein-coding gene described in D2 is the CRISPR-Cas9 system, which contains gRNA and Cas9 targeting the Lupus La protein-coding gene. The substance that targets the Lupus La protein-coding gene described in D2 is a gRNA that targets the Lupus La protein-coding gene. The sequence of the gRNA targeting the Lupus La protein-encoding gene is shown in SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No.

7.

7. The application according to claim 6, characterized in that: The drug described in D2 that reduces the efficiency of activation-induced cytidine deaminase targeting immunoglobulin heavy chain loci is a drug for treating diseases with increased γH2AX focal points, or a drug for treating or / and diseases with immunoglobulin heavy chain-Myc translocations.

8. A method for promoting antibody class switching rearrangements and / or high-frequency somatic mutations in B cells, characterized in that, The method includes introducing the Lupus La protein or its encoding gene as described in any of claims 1-3 into target B cells to promote antibody class switching rearrangement and / or somatic high-frequency mutation steps in the target B cells.

9. Recombinant B cells, characterized in that, The recombinant B cells are derived from target B cells and are prepared by a method comprising the following steps: introducing the Lupus La protein or its encoding gene as described in any one of claims 1-3 into target B cells to obtain recombinant B cells.

10. A method for shortening the antibody development cycle, characterized in that, include: 1) Providing cells expressing the Lupus La protein as described in any one of claims 1-3; 2) Contact the cells described in step 1) with antigen-specific B cells to enhance the somatic high-frequency mutation frequency of the antigen-specific B cells, thereby shortening the antibody development cycle.

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